文献类型：期刊文献

中文题名：大肠杆菌胸苷激酶基因的克隆表达与应用研究

英文题名：Cloning,Expression and Application of Thymidine Kinase from Escherichia coli K-12

作者：张广宇[1];丁庆豹[1];欧伶[1]

机构：[1]华东理工大学生物工程学院,上海200237

年份：2013

卷号：23

期号：5

起止页码：20

中文期刊名：生物技术

外文期刊名：Biotechnology

收录：北大核心:【北大核心2011】；CSCD:【CSCD_E2013_2014】；

语种：中文

中文关键词：胸苷;胸苷酸;胸苷激酶

外文关键词：Thymidine ; Thymidylate ; Thymidine kinase

摘要：目的:构建含大肠杆菌胸苷激酶基因(tdk)的重组质粒,并对胸苷激酶性质进行研究。方法:通过PCR技术从大肠杆菌K-12中扩增出tdk基因,并克隆到表达载体pET-28a(+)上,转入大肠杆菌BL21(DE3)中构建重组菌株(DTK)。结果:成功扩增出了tdk基因,并构建了重组菌株。结论:获得的tdk基因长为618bp,重组菌株能表达出大量可溶性胸苷激酶蛋白。胸苷激酶的酶比活力可达到1 600U/mg;酶最适温度为45℃;最适pH为8.0。同时,胸苷激酶最佳反应条件为:50mmol/L pH 8.0的Tris-HCl缓冲液,30U的粗酶,5mmol/L的MgCl2,5mmol/L的TR,5mol/L的ATP,45℃反应100min,胸苷酸的转化率可达到60%以上。
Objective：Construct the recombinant plasmid containing the gene encoding thymidine kinase from Escherichia coli K - 12 and study the properties of thymidine kinase. Method：The sequence of tdk gene was amplified by PCR from Escherichia coli K - 12 and was cloned into expression vector pET-28a（ ＋ ）, then the recombinant plasmid was transformed into the strain E. coli BL1 （ DE3 ）. Result： The tdk gene was amplified and the recombinant strain was constructed successfully. Conclusion：The tdk gene was 618 bp in length and thymidine kinase was highly expressed. The specific activity of thymidine kinase reached to 1 600U/mg. The optimum temprature was 45℃ and pH was 8. 0. The optimum condition of reaction catalyzed by thymidine kinase was 50mmolZL Tris - HCI （pH 8. 0）, 30U enzyme, 5mmolZL MgC12 ,Smmol/L TR,SmolZL ATP. The conversion rate of thymidylate could reach to 60% after 100 min at 45℃.